UNLABELLED: Poor ocular bioavailability of drugs (<1%) from conventional eye drops (ie, solution, suspension, and ointments) is mainly due to the physiologic barriers of the eye. In general, ocular efficacy is closely related to ocular drug bioavailability, which may be enhanced by increasing corneal drug penetration and prolonging precorneal drug residence time. In our current work, we develop and evaluate a new colloidal system, that is, poly(dl-lactide-co-glycolide) (PLGA) nanoparticles for sparfloxacin ophthalmic delivery, to improve precorneal residence time and ocular penetration. Nanoparticles were prepared by nanoprecipitation technique and characterized for various properties such as particle size, zeta potential, in vitro drug release, statistical model fitting, stability, and so forth. Microbiological assay was carried out against Pseudomonas aeruginosa using the cup-plate method. Precorneal residence time was studied in albino rabbits by gamma scintigraphy after radiolabeling of sparfloxacin by Tc-99m. Ocular tolerance of the developed nanosuspension was also studied by the Hen Egg Test-Chorioallantoic Membrane (HET-CAM) method. The developed nanosuspension showed a mean particle size in the range of 180 to 190 nm, suitable for ophthalmic application with zeta potential of -22 mV. In vitro release from the developed nanosuspension showed an extended release profile of sparfloxacin according to the Peppas model. Acquired gamma camera images showed good retention over the entire precorneal area for the developed nanosuspension compared with that of a marketed formulation. The marketed drug formulation cleared very rapidly from the corneal region and reached the systemic circulation through the nasolacrimal drainage system, as significant radioactivity was recorded in kidney and bladder after 6 hours of ocular administration, whereas the developed nanosuspension cleared at a very slow rate (P < .05) and remained at the corneal surface for longer duration, as no radioactivity was observed in the systemic circulation. HET-CAM assay with 0 score in 8 hours indicates the nonirritant property of the developed nanosuspension. The developed lyophilized nanosuspension was found to be stable for a longer duration of time than the conventional marketed formulation with a good shelf life. FROM THE CLINICAL EDITOR: Poor ocular bioavailability of drugs (<1%) from conventional eye drops is mainly due to the eye physiological barriers. In this study, a new colloidal system, PLGA nanoparticle for sparfloxacin ophthalmic delivery was demonstrated to improve precorneal residence time and ocular penetration. The developed lyophilized nanosuspension was found to be stable for longer duration of time than conventional marketed formulations.